WO2002057490A9 - Procedes permettant d'associer des caracteres quantitatifs a des alleles chez des paires d'enfants de memes parents - Google Patents

Procedes permettant d'associer des caracteres quantitatifs a des alleles chez des paires d'enfants de memes parents

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Publication number
WO2002057490A9
WO2002057490A9 PCT/US2001/045459 US0145459W WO02057490A9 WO 2002057490 A9 WO2002057490 A9 WO 2002057490A9 US 0145459 W US0145459 W US 0145459W WO 02057490 A9 WO02057490 A9 WO 02057490A9
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population
sib
sibling
phenotypic
method described
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PCT/US2001/045459
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WO2002057490A3 (fr
WO2002057490A2 (fr
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Joel S Bader
Aruna Bansal
Pak Sham
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Curagen Corp
Joel S Bader
Aruna Bansal
Pak Sham
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Application filed by Curagen Corp, Joel S Bader, Aruna Bansal, Pak Sham filed Critical Curagen Corp
Publication of WO2002057490A2 publication Critical patent/WO2002057490A2/fr
Publication of WO2002057490A9 publication Critical patent/WO2002057490A9/fr
Publication of WO2002057490A3 publication Critical patent/WO2002057490A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection

Definitions

  • the invention relates to a method for detecting an association in a population of individuals between a genetic locus or loci and a quantitative phenotype.
  • Linkage analysis a traditional method for identifying the genes responsible for a monogenic disorder by identifying genetic markers in linkage disequilibrium with a particular phenotype, loses power for complex disorders because no single disease-related gene is expected to have large penetrance.
  • a more recent approach is to search for alleles whose inheritance is associated or correlated with changes in a phenotypic value.
  • Single nucleotide polymorphisms, or SNPs can provide such a marker set. These are typically bi-allelic markers with linkage disequilibrium extending an estimated 10,000 to 100,000 nucleotides in heterogeneous human populations. Tens to hundreds of thousands of these closely-spaced markers are required for a complete scan of the 3 billion nucleotides in the human genome. Because each SNP constitutes a separate test, the significance threshold must be adjusted for multiple hypotheses (p-value ⁇ 10 "8 ) to identify statistically meaningful associations. Consequently, hundreds to thousands of individuals are required for association studies.
  • the population size required for an association test may be reduced by limiting the effect of confounding factors, such as environmental effects or spurious association with markers correlated with ethnicity.
  • Case-control studies which are used to increase the homogeneity of a test population for studies of diseases with a clear distinction between affected and unaffected, are less applicable to quantitative phenotypes.
  • An alternative has been to conduct genetic studies in highly homogenous populations. A drawback of this approach, however, is that disease- associated markers or causative alleles found in an isolated population might not be relevant for a larger population.
  • a second and more attractive alternative is to use a test population composed of siblings.
  • An alternative that circumvents the need for individual genotypes, related to previous DNA pooling methods for determination of linkage between a molecular marker and a quantitative trait locus, is to determine allele frequencies for sub-populations pooled on the basis of a qualitative phenotype. Populations of unrelated individuals, separated into affected and unaffected pools, have greater power than related populations. If a population consists of sib-pairs, concordant pairs versus unrelated controls have greater power than discordant pairs separated into affected and unaffected pools. Nevertheless, discordant designs might provide a better control for confounding factors such as age, ethnicity, or environmental effects.
  • the phenotypes relevant for complex disease are often quantitative, however, and converting a quantitative score to a qualitative classification represents a loss of information that can reduce the power of an association study.
  • the location of the dividing line for affected versus unaffected classification, for example, can affect the power to detect association.
  • Furthemiore, pooling designs based on a comparison of numerical scores are not even possible with a qualitative classification scheme. These distinctions can be especially relevant when populations contain related individuals and qualitative tests have a disadvantage.
  • the invention provides a method for detecting an association in a population of individuals between a genetic locus and a quantitative phenotype, wherein two or more alleles occur at the locus, and wherein the phenotype is expressed using a numerical phenotypic value whose range falls within a first numerical limit and a second numerical limit.
  • the method includes: (a) obtaining the phenotypic value for each individual in the population; wherein said population comprises sibling pairs; (b) selecting a first subpopulation of individuals having phenotypic values that are higher than a predetermined lower limit and pooling DNA from the individuals in the first subpopulation to provide an upper pool; (c) selecting a second subpopulation of individuals having phenotypic values that are lower than a predetermined upper limit and pooling DNA from the individuals in the second subpopulation to provide a lower pool; (d) for one or more genetic loci, measuring the difference in frequency of occurrence of a specified allele between the upper pool and the lower pool; and (e) determining that an association exists if the allele frequency difference between the pools is larger than a predetermined value.
  • the phenotypic value is obtained as a numerical combination of other phenotypic values.
  • the phenotypic value can be obtained from regressing out the effect of age.
  • the phenotypes are numerical rankings.
  • the lower limit and the upper limit are chosen such that, for a specified false- positive rate, the frequency of occurrence of false-negative errors is minimized.
  • the population comprises unrelated individuals.
  • the predetermined lower limit is set so that the upper pool includes the highest 35% of the population and the predetermined upper limit is set so that the lower pool includes the lowest 35% of the population.
  • the predetermined lower limit is set so that the upper pool includes the highest 30% of the population and the predetermined upper limit is set so that the lower pool includes the lowest 30% of the population.
  • the predetermined lower limit is set so that the upper pool includes the highest 27% of the population and the predetermined upper limit is set so that the lower pool includes the lowest 27% of the population.
  • each family is considered as a unit, and either (i) both sibs are selected for the upper pool; (ii) both sibs are selected for the lower pool; or (iii) neither sib is selected.
  • selection is based on the mean phenotype of the two sibs. Selection can be based on both sibs being above a threshold or below a threshold.
  • the individuals in the population are sibling pairs and each pair is ranked according to a mean value of the phenotypic values of the siblings in each pair, and for sibling pairs that are in a pool, both members of the sibling pair are in the same pool.
  • the predetermined lower limit is set, e.g., so that the upper pool includes the pairs with highest 35% of the mean values in the population and the predetermined upper limit is set so that the lower pool includes the lowest 35% of the mean values in the population.
  • the predetermined lower limit is set so that the upper pool includes the highest 30% of the mean values in the population and the predetermined upper limit is set so that the lower pool includes the lowest 30% of the mean values in the population, or the predetermined lower limit is set so that the upper pool includes the highest 27% of the mean values in the population and the predetermined upper limit is set so that the lower pool includes the lowest 27% of the mean values in the population.
  • each sib-pair is considered as a unit, and either (i) one sib is selected for the upper pool, and the other sib is selected for the lower pool; or (ii) neither sib is selected.
  • selection is based on the magnitude difference between sib phenotype values. For example, selection can be based on one sib being above a threshold and the other sib being below a threshold.
  • the individuals in the population are sibling pairs
  • the pairs are ranked by the absolute magnitude of the difference in phenotypic value for the sibs within each pair, the percent of pairs with greatest difference are identified, the percent of pairs being 70%, and the siblings in each pair are distributed such that the sibling with the high phenotypic value is selected for the upper pool and the sibling with the low phenotypic value is selected for the lower pool, providing 35% of the population in each pool.
  • the percent of pairs can be about 60% and the distribution provides 30% of the population in each pool. In another example, the percent of pairs is 54% and the distribution provides 27% of the population in each pool.
  • the individuals in the population are sibling pairs
  • the results can be obtained using, e.g., family tests for sib-pairs.
  • Each family is considered as a unit, and either (i) both sibs are selected for the upper pool; (ii) both sibs are selected for the lower pool; or (iii) neither sib is selected.
  • each sib-pair is considered as a unit, and either (i) one sib is selected for the upper pool, and the other sib is selected for the lower pool; or (ii) neither sib is selected.
  • an unrelated population is selected from a sib-pair population and pooling is conducted on the derived unrelated population, hi some preferred embodiments, the sibling with phenotype furthest from the overall mean is selected, from each family to generate an unrelated population.
  • unrelated individuals are provided by a process comprising the steps of: (a) providing a superpopulation of individuals, each individual being a member of a sibling pair; (b) selecting that member of each sibling pair having a phenotypic value such that the absolute value of the difference between the individual's phenotypic value and either the first numerical limit or the second numerical limit is lower than the difference for the other individual in the pair, thus providing a population of unrelated individuals; (c) setting the predetermined lower limit so that the upper pool includes the highest 36% of the population and the setting the predetermined upper limit so that the lower pool includes the lowest 36% of the population.
  • one member of each sibling pair is chosen at random to provide a group of unrelated individuals; and the members of the group having phenotypic values greater than a predetermined lower limit are placed in the first subpopulation and the members of the group having phenotypic values lower than a predetermined upper limit are placed in the second subpopulation.
  • only one member of a sibling pair is placed in a subpopulation; wherein the fraction of individuals in the first subpopulation is determined using Equation A and the fraction of individuals in the second subpopulation is determined using Equation B, and wherein the sibling with genotype G ⁇ is selected for the upper pool if the value of ⁇ is in the interval 0 ⁇ ⁇ ⁇ n/2 or is selected for the lower pool if the value of ⁇ is in the interval ⁇ ⁇ 3 ⁇ d2 and the sibling with genotype G is selected otherwise.
  • the mean phenotypic value for the pair is calculated; and the first subpopulation contains those pairs whose mean phenotypic value is greater than a predetermined mimmum value and the second subpopulation contains those pairs whose mean phenotypic value is lower than a predetennined maximum value.
  • the difference between the phenotypic values for the members of each sibling pair is calculated; (ii) those sibling pairs whose values of the calculated difference are greater than a predetermined minimum value for the difference are identified; and (iii) in each identified sibling pair, placing the sibling with the higher phenotypic value in the first subpopulation and the sibling with the lower phenotypic value in the second subpopulation.
  • the mean phenotypic value for the pair is calculated; and (ii) a first upper subpopulation contains those pairs whose mean phenotypic value is greater than a predetermined minimum value and a first lower subpopulation contains those pairs whose mean phenotypic value is lower than a predetermined maximum value; (iii) the difference between the phenotypic values for the members of each sibling pair is calculated; (iv) those sibling pairs whose values of the calculated difference of step (iii) are greater than a predetermined minimum value for the difference are identified; and (v) in each sibling pair identified in step (iv), placing the sibling with the higher phenotypic value in a second upper subpopulation and the sibling with the lower phenotypic value in a second lower subpopulation.
  • Fig. 1 The population size required to detect association with a test of pooled DNA is shown as a function of the fraction of population p selected for each pool, relative to the population size required for a regression test using individual genotyping, for a QTL making a small contribution to a complex trait.
  • the same family structure and the same phenotypic variable, either the individual phenotype, the sib-mean, the sib-difference, or the combined results from sib-mean and sib-difference tests, are used for tests based on pooling and individual genotyping. All of these tests show the same relative efficiency as a function of pooling fraction, with an optimal fraction of 0.27 requiring only 1.24x the population for individual genotyping.
  • Fig. 2 The population size required to detect association for the sib-radial design, relative to the population required for a combined regression test using individual genotypes, is shown as a function of the sibling phenotypic correlation t R .
  • Fig. 3 The number of individuals required for pooling designs with a sib-pair family structure is compared to the number of unrelated individuals for an association test of equivalent power and significance as a function of the sibling phenotypic correlation t R .
  • sib-radial referred to as Mahanlobis
  • sib-mean is a dashed line
  • sib-difference is a dot-dashed line
  • sib-combined is a thick line.
  • Fig. 5 Exact numerical results for the population size required to detect association is shown as a function of the allele frequency * for (A) dominant inheritance, (B) additive inheritance, and (C) recessive inheritance for tests using pooled DNA.
  • the variance ratio OA / ⁇ ?e is 0.02
  • the type I error is 5xl0 -8
  • the type II error is 0.2
  • the pooling fraction 0.27 is used for all designs except sib-radial, for which 0.188 is used.
  • the population required to detect association with specified power and significance is almost flat asp decreases below 0.5, then rises sha ⁇ ly as p falls below a critical value dependent on inheritance mode, approximately ⁇ lS for a dominant inheritance, cr /2 for additive inheritance, and cr 3 /2 for recessive inheritance.
  • sib-radial design is less robust to small allele frequencies than the other designs, h this figure, unrelated- population is a dotted line, sib-radial (referred to as Mahanlobis) is a thin line, sib-mean is a dashed line, sib-difference is a dot-dashed line, and sib-combined is a thick line.
  • Mahanlobis sib-radial
  • sib-mean is a dashed line
  • sib-difference is a dot-dashed line
  • sib-combined is a thick line.
  • Pi frequency of allele A ⁇ in sib i either 1, 0.5, or 0 for an autosomal marker
  • N the total number of individuals whose DNA is available for pooling n number of individuals selected for a single pool p pooling fraction defined as n/N
  • PV-P L frequency of allele A ⁇ in the upper (U) or lower (L) pool T test statistic which is expected to be close to zero when the genotype G does not affect the phenotypic value and is expected to be non-zero when individuals with genotypes A ⁇ A ⁇ , A ⁇ A 2 , and ⁇ 4 2 ⁇ 4 2 have different mean phenotypic values.
  • T> z a corresponds to statistical significance at level a, typically termed ap-valne.
  • a typical threshold for significance is ap- value smaller than 0.05 or 0.01. If M independent tests are conducted, a conservative correction that yields a finals-value of is to use ap-value of ⁇ JM fox each of the tests. ⁇ type II error rate (false-negative rate). The power of a test is 1- ⁇ .
  • sibling relationship when two individuals are "related to each other", they are genetically related in a direct parent-child relationship or a sibling relationship. In a sibling relationship, the two individuals of the sibling pair have the same biological father and the same biological mother.
  • sib is used to designate the word “sibling”, and the sibling relationship is defined above.
  • sib pair is used to designate a set of two siblings.
  • the members of a sib pair may be dizygotic, indicating that they originate from different fertilized ova.
  • a sib pair includes dizygotic twins.
  • gene or related terms, including alleles that may occur at a particular genetic locus.
  • a focus of the present invention is to examine the statistical power of pooling designs for quantitative phenotypes.
  • Five basic types of pooling designs are considered: selecting unrelated individuals, selecting sib pairs for the same pool, selecting sib pairs and splitting each pair between pools in two different ways, and a combined test of the sib-together and sib-apart tests.
  • the selection rules are optimized to minimize the population requirements for each type of design, and the powers of the designs are compared with each other and with individual genotyping.
  • a variance components model is used as the basis for the optimization of pooling designs.
  • This model describes the joint phenotype-genotype distribution of an unselected population and includes a term specifically describing the phenotypic correlation between siblings.
  • the test statistic used to detect association is the allele frequency difference between two pools.
  • Analytical formulas for population size requirements are derived for a QTL making a small contribution to the phenotypic values, exactly the limit that attains for a complex trait. The validity of the formulas is ascertained by exact numerical computation over a wide range of parameters of the population model, including the effect of the QTL, the frequency of the minor allele and its dominant, recessive, or additive inheritance mode. The sensitivity of the results to the genetic model and the pooling design are also explored with exact numerical computation.
  • a standard variance components model is used to describe the joint phenotype-genotype probability distribution (Falconer and MacKay 1996).
  • a quantitative phenotype X standardized to mean 0 and variance 1, is hypothesized to be affected by the genotype G at a biallelic locus with minor allele A ⁇ and major allele A 2 occurring at population frequencies/? and 1-p. More generally, A 2 may represent any of a number of alternate alleles, and 1-p their aggregate frequency.
  • the population is assumed to be random mating and in Hardy- Weinberg equilibrium.
  • the symbol P is used to denote a probability, and the genotype frequencies P(G) are/?
  • the frequency of allele ⁇ ! in genotype G denoted pc is 1 for A ⁇ A ⁇ , 0.5 for A ⁇ A 2 , and 0 for A 2 A .
  • the variance of the allele frequency per individual is/?(l-/?)/2 and is denoted ⁇ p 2 .
  • the genotype combination for a sib pair is denoted P(G ⁇ ,G 2 ).
  • the probability distribution of the 9 possible combinations of dizygotic sib-pair genotypes, shown in Table I, can be derived by considering all possible parental mating types and their offspring genotype distributions (Neale and Cardon 1992).
  • the genetic correlation between sibs implies that P(G h G 2 ) ⁇ P(G ⁇ )P(G 2 ).
  • the effect ⁇ (G) of genotype G on the phenotype is a- ⁇ , d- ⁇ , and - ⁇ - ⁇ for genotypes A ⁇ A- i, A ⁇ A 2 , and A 2 A 2 respectively.
  • the constant ⁇ - ⁇ (2p - 1) + 2dp(l-p) ensures that the phenotype has mean 0.
  • the ratio dl ⁇ determines the inheritance mode of allele A ⁇ .
  • the ratio is -1 for a recessive allele, +1 for a dominant allele, and 0 for an additive allele.
  • Co-dominance implies a ratio between -1 and +1, while over-dominance implies a ratio with a magnitude greater than 1.
  • O R 2 1- • 9 9 O + ⁇ b
  • o ⁇ + ⁇ o are small for any particular QTL, and oft is close to 1.
  • the additive variance is typically larger than the dominance variance, even for alleles with a dominance or recessive inheritance mode.
  • the probability density of phenotypic values for sib pairs isf(X ⁇ [ 2 ), where the symbol/is used generally to denote a probability density.
  • t and t R are nearly identical. The are normalized to 1 for integration overXi and 2 .
  • X ⁇ and 2 are a natural choice of coordinates for expressing sibling phenotypic values, it is more convenient to introduce other coordinate systems in which the variables are uncorrelated and the probability distributions are separable when the QTL has a small effect.
  • the variance of the sib-mean and sib-difference variables may be expressed more generally as
  • R ⁇ [l ⁇ (s-l)r]/s
  • the family size s is 2 for sib-pairs
  • the genotypic correlation r is 1/2 for full sibs.
  • Radial coordinates are a second choice yielding a separable probability distribution in the absence of the QTL.
  • the radial distance b and phase ⁇ are defined so that
  • Contour lines of equal probability density in the X X 2 plane are ellipses tilted at 45° with a ratio of major axis to minor axis of [(l+t)/(l-t)] 1/2 .
  • N individuals are unrelated, a particularly simply design is to select the n individuals whose phenotypic values are at the upper and lower tails of the distribution, which defines upper and lower thresholds Xu and X L .
  • This design has been analyzed previously in the context of both selection (Ollivier 1997) and association (Bader et al. 2000) studies and is here termed the unrelated-population design.
  • a corresponding design for sib pairs is termed unrelated-random.
  • one sib is chosen at random from each sib-ship to generate a population of N/2 unrelated individuals. Individuals at the upper and lower tails of this unrelated population are then selected for pooling.
  • the unrelated-random design for N individuals with pooling fraction p is exactly equivalent to the unrelated-population design for N/2 individuals with pooling fraction 2p.
  • the unrelated-random design is not expected to be as powerful as sib-pair methods making use of family structure information.
  • a second design selecting only unrelated individuals is termed sib-radial.
  • n sib-ships with the largest magnitude b and a positive sib-mean X + are identified, and the sibling with the larger phenotypic value is selected for the upper pool.
  • the n sib- ships with the largest b and negative sib-mean are identified, and the sibling with the more negative phenotypic value is selected for the lower pool.
  • the sib-mean design selects each sib-ship as a family unit based on the phenotypic mean of the pair.
  • the n/2 pairs at the extreme upper and lower tails of the distribution of phenotypic means for sib-ships, comprising n individuals each, are selected for the upper and lower pools.
  • the upper and lower thresholds are again termed Xu and X L .
  • the sib-difference design selects individuals based on the difference of phenotypic values within each sib-ship, or equivalently the within-family phenotypic variance. The n sib-pairs with the greatest variance are identified. Within each family, the individual with the higher phenotypic value is selected for the upper pool, and the individual with the lower phenotypic value is selected for the lower pool.
  • the threshold for the magnitude of the difference is termed X ⁇ .
  • sib-mean and sib-difference methods are similar to between-family and within-family selection methods for breeding value (Falconer and MacKay 1996), with two notable differences.
  • Within- family selection is typically applied to large family sizes without reducing the number of families, while here the number of families is reduced according to within-family variance.
  • individuals are selected here both for extreme high and extreme low phenotypic values rather than for values extreme in only one direction. Breeding methods concerned with only a single direction are closer analogs to concordant and discordant selection based on affected/unaffected status (Risch and Teng 1998).
  • the results of the sib-mean and sib-pair design may be combined as a single, more powerful test, as is commonly done with regression tests for individual genotyping (Abecasis 2000).
  • the test significance for an association study based on pooled DNA is the difference ⁇ p in allele frequency between the frequencies pu and/? , measured for each pool.
  • ⁇ p has a normal distribution with variance defined as ⁇ 0 2 / « under the null hypothesis and ⁇ 2 ln under the alternative hypothesis.
  • ⁇ L (G) p- l ⁇ [X L - ⁇ (G)]/ ⁇ R ⁇ P(G).
  • Nunrel-ran 2[(2p)l2y 2p ] (ZorZ ⁇ - ⁇ ) ⁇ R 2 / ⁇ A , twice as large as N ur ⁇ r ei-popi n with a pooling fraction half as large.
  • thresholds b v and b L are established for the upper and lower pool by the normalization equations
  • the factor of (1/2) arises because only one sib is selected from each sib-ship, and we have used the approximation that the QTL makes a small contribution to the phenotype correlation t.
  • the sibling with genotype G 1 is selected for the upper pool in the interval 0 ⁇ ⁇ ⁇ l2 and for the lower pool in the interval ⁇ ⁇ 3 ⁇ /2; the sibling with genotype G 2 is selected otherwise.
  • ⁇ L (G) p- ⁇ ⁇ ⁇ P(G,G') ⁇ d ⁇ jdb b f(b, ⁇ G,G'), ⁇ where the symmetry between siblings has allowed the change in integration limits for ⁇ to consider only the regions where sibling 1 is selected. Numerical results for the required population size may then be obtained as outlined above for the unrelated-population design.
  • the expected allele frequencies in the upper and lower pools are
  • E(pu, L ) P ⁇ [(2 bj ⁇ ) + ⁇ (-b p )lp(2 ⁇ ) x/2 ] [ R + /T + xa + RJT ' 2 ] ⁇ p ⁇ A l ⁇ R , where the upper pool has the positive deviation from/? and the lower pool the negative deviation.
  • Nsib-radiai (2pt [(2b J ⁇ ) + ⁇ (-b p )l p(2 ⁇ ) m ] ⁇ 2 [ R + IT + m + RJT 12 ] ⁇ 2 (z a -z ⁇ ) 2 ⁇ R 2 l ⁇ A 2 .
  • Nsib-ra d iai 2.90 [ R + IT + X/2 + RJT- ⁇ n ] ⁇ 2 (z a -z ⁇ _ ⁇ f ' ⁇ R 2 ' ⁇ / ' ⁇ A 2 for this pooling design.
  • the subsequent factor depends only on the phenotypic correlation t R between siblings.
  • ⁇ u(G ⁇ ,G 2 ) p- ⁇ ⁇ -[Xu- ⁇ (G)]l ⁇ R ⁇ P(G ⁇ ,G 2 )
  • ⁇ L (G ⁇ ,G 2 ) p ⁇ l ⁇ [X L - ⁇ (G)]l ⁇ R ⁇ P(G ⁇ ,G2).
  • G 1 > G 2 and ? + (G 1? G 2 ) the sib-mean allele frequency as defined previously.
  • G ⁇ ,G 2 ⁇ 2 s ⁇ ⁇ [ ⁇ u(G ⁇ ,G 2 ) + ⁇ L (G ⁇ ,G 2 )] [p + (G ⁇ ,G 2 ) ⁇ 2 ⁇ -s(pu 2 +p L 2 ).
  • the factor s 2 accounts for the family structure, as nls rather than n measurements of p + are averaged to determine the allele frequency of each pool.
  • the variance under the null hypothesis may be derived directly from the sib-pair genotype frequencies, or more simply by noting that the variance of the mean allele frequency for a sib-pair is R + ⁇ p 2 , which is (3/4) as large as the variance ⁇ p 2 for an individual.
  • the variance for each pool is reduced by averaging over n/2 such terms, and multiplying by 2 for the number of pools yields 3 ⁇ p .
  • Nsib-mean (s ⁇ l2y p ) (T+IR+) (z a -z ⁇ - ⁇ ) 2 ⁇ R l ⁇ A 2 .
  • the factor ply p 2 is optimized with a pooling fraction of 0.27, yielding
  • Nsi -mean 2.47 (T+/R+) (z a -z ⁇ - ⁇ f ⁇ R 2 l ⁇ A 2 for the corresponding population size.
  • a sib pair is selected if the sib-difference X_ is larger in magnitude than a threshold Xr,
  • sibling 1 has the higher phenotype and is selected for the upper pool, and sibling 2 is selected for the lower pool.
  • the roles of the siblings are reversed.
  • G l t G 2 ⁇ , 2 2 ⁇ [2 ⁇ u(G ⁇ ,G 2 ) + 2 ⁇ L (G ⁇ ,G 2 )]p 2 - ( ⁇ p) 2 .
  • ⁇ 0 2 The value of ⁇ 0 2 under the null hypothesis may be obtained more simply by noting that the allele frequency difference between two siblings has variance ⁇ p , and the measured allele frequency difference is the average of n such terms.
  • the population size required to detect association may be determined exactly by numeric calculation of the threshold value X T as a function of the pooling fraction p. This value is then
  • An analytic expression accurate when ⁇ R is close to 1 may be derived using the same technique as for the previous pooling designs.
  • Nsib-diff (spl2y 2 )(TJR-)(z a -z ⁇ - ⁇ ) 2 ⁇ R 2 l ⁇ A 2 .
  • N s ib-diff 2.47 (TJI )(z a -z ⁇ - ⁇ ) 2 ⁇ R l ⁇ A 2 is the corresponding population size.
  • sib-mean and sib-difference design Because the sib-mean variables X + and/? + are uncorrelated from the sib-difference variables X_ and ?-, association tests based separately on these sets of variables are statistically independent and may be combined to achieve greater power even when the same unselected population is used and even when the same sib pairs are selected under both designs.
  • the combined test uses the measured value of ⁇ p as an estimator for ⁇ A l ⁇ R ,
  • E( ⁇ A / ⁇ R ) (T ⁇ x/2 /R ⁇ )(p/2y p ⁇ p )E( ⁇ p), with the + sign for the sib-mean test and the - sign for the sib-difference test.
  • the variance of the estimator is the variance of ⁇ p, obtained previously as 2sR ⁇ p 2 ln, multiplied by the square of the preceding terms, or
  • the sib-mean and sib-difference estimators are summed with weights proportional to the inverse of the estimator variance.
  • the variance of the combined estimator is
  • Ncomb (sp!2y 2 ) [(R + /T + ) + (RJT.)Y X ⁇ R 2 l ⁇ A 2 .
  • the prefactor (spl2y p ) is 2.47. Since the variance of the individual estimators are identical under the null and alternative hypothesis, the population size for the combined estimator is simply the reciprocal of the sum of the reciprocal population sizes required for the individual estimators.
  • X t m(pt- ⁇ ) + £i where i labels an observation, X,- is an observed phenotypic variable with mean 0,p t is a observed genotypic variable with mean/?, and Si is the residual contribution not explained by the model.
  • the phenotype and genotypic variables for a regression test are the individual X t and ?, values measured for N unrelated individuals and the sib-mean and sib-difference variable X ⁇ and p ⁇ for N/2 sib-pairs.
  • An estimator formed by combining the sib-mean and sib-difference estimators has a population size requirement of
  • N regr s[R + /T + + RJT r x (z a -z ⁇ - ⁇ ) 2 ⁇ R 2 / ⁇ A 2 .
  • Results for required population sizes were obtained numerically using computations converged to 1 part in 10 6 (Press et al. 1997).
  • Brent's root-finding algorithm was used to determine the threshold values Xu andXz for the upper and lower pools for a given pooling design and pooling fraction p;
  • Brent's optimization algorithm was then used to find the p with maximum power. While the reported results are based on a normal approximation for the allele frequency difference ⁇ p, results were also obtained for the exact multinomial distribution for the unrelated- population design. The difference between the numerical results for the multinomial and normal distributions was typically less than 1%.
  • the population size required for the pooling combined estimator was obtained numerically as the reciprocal of the sum of the reciprocal exact sizes required for the sib-mean and sib-difference pooling designs. Using a 750 MHz Pentium III running Linux, the root-finding and minimization for each parameter set required less than 0.01 sec each design.
  • the population size requirements of pooled DNA methods are shown in Fig. 1 relative to corresponding regression tests for the same family structure for the unrelated-population, sib- mean, sib-difference, and combined designs, as well as the sib-radial design which will be discussed separately below.
  • the ratio of population size requirements is independent of all model parameters except for the fraction p of individuals whose DNA is pooled. Furthermore, the ratio is independent of family structure for these matched comparisons.
  • the pooling fraction p 0.27 is seen to be optimal. The curve is flat near the minimum, indicating that pooling fractions close to 0.27 give near-optimal results. For these designs, population sizes must be increased by 1.24x to attain the same power as would have been achieved with N individual genotypes.
  • the performance of the sib-radial design relative to the combined regression test for individual genotypes is shown as a function of the residual sibling phenotypic correlation t R , with the optimal fraction 0.188 used to construct the upper and lower pools.
  • the ratio of population sizes is roughly 1.5 until the phenotypic correlation rises above 0.6, at which point the population size requirements for the sib-radial design begin to rise more steeply.
  • Fig. 3 the population size requirements for association tests using DNA pooled from sib pairs are shown as a function of the residual sibling phenotypic correlation t , relative to the population size required for a test of DNA pooled from unrelated individuals. Ratios larger than 1 indicate that the population of N unrelated individuals is more powerful than a population of N/2 sib pairs, while ratios smaller than 1 indicate that the sib-pair population is more powerful. These ratios are derived from the appropriate analytic expressions in the limit of a QTL making a small contribution to a complex trait.
  • the combined test using sib-mean and sib-difference pools is uniformly the most powerful sib-pair design for all values of t R . Its worst-case performance relative to an unrelated population occurs when t R is (3 1/2 +l)/(3 1/2 -l), or 0.2679, where it requires a population 7% larger.
  • the unrelated and sib-pair tests require the same population size when the phenotypic correlation is 0.5, and the sib-pair test becomes much more powerful for equal population sizes for larger values oft ⁇ .
  • sib-pair designs requiring only a single pair of pools, a population of unrelated individuals is more powerful than a population of sib pairs except for large values of the sibling phenotypic correlation, t R > 0.75, at which point the sib-radial and sib-difference designs become more powerful. Below this phenotypic correlation, the sib-radial design is substantially more powerful than the other sib-pair tests; above this correlation, the sib-difference design is only slightly more powerful than the sib-radial design. As t R increases, the sib-mean design decreases in power and the sib-difference design increases in power. 3.3 Sensitivity to QTL contribution, allele frequency, inheritance mode
  • the population size requirements for pooling tests is inversely proportional to the additive variance contributed by the QTL relative to the residual phenotypic variance, ⁇ A l ⁇ R , and independent of any remaining parameters of the genetic model.
  • ⁇ A l ⁇ R residual phenotypic variance
  • the type I error rate a is 5x 10 -8 and the type II error rate /?is 0.2; these values have been suggested for whole-genome scans (Risch and Merikangas 1996).
  • the sibling phenotypic correlation t R was also held fixed for the numerical tests.
  • Estimates of the genetic heritability for complex traits range from 20% for cancer (Verkasalo et al. 1999), 20% to 40% for Type 2 diabetes mellitus (NTDDM) (Watanabe et al. 1999), 50% for pulmonary function (Wilk et al. 2000), 10% to 50% for systolic and diastolic blood pressure (Iselius et al.
  • Fig. 4A the population size required by each pooling design is shown as a function of ⁇ A l ⁇ R , the QTL additive variance relative to the residual phenotypic variance l- ⁇ - ⁇ D 2 .
  • the QTL has purely additive inheritance and the minor allele frequency is 0.1.
  • the minor allele frequency of 0.1 used in this example is typical for polymorphisms in coding regions. Reported minor-allele frequencies for S ⁇ Ps found in multiple populations range from 0.05 to 0.25, with lower frequencies for variations which cause non-conservative amino acid changes and higher frequencies for conservative substitutions and changes in non-coding regions (Cargill et al. 1999, Goddard et al. 2000).
  • the unrelated-population design is a dotted line
  • sib- radial is a thin line
  • sib-mean is dashed
  • sib-difference is dot-dashed
  • the combined estimator sib-comb is a thick line.
  • the sib- radial design is less powerful than predicted by analytic theory for ⁇ A l ⁇ R 2 > 0.05, roughly the transition between a complex trait and a monogenic trait.
  • the allele frequency difference at the significance threshold, z a ⁇ ln xl2 is shown in Fig. 4B for the same set of parameters. As the QTL contribution is smaller, allele frequency differences must be measured with precision. While raw frequency differences of 10% are significant for a monogenic trait, ⁇ A l ⁇ R 2 ⁇ 0.1, raw frequencies differences of 3% must be measured with little error to achieve maximum power for a complex trait with ⁇ A l ⁇ R ⁇ 0.01.
  • Figs. 5 and 6 The sensitivity of the results to both the allele frequency ? and the inheritance mode dla is shown in Figs. 5 and 6.
  • the pooling fractions are fixed at the limiting values 0.27 for the unrelated-population, sib-mean, sib-difference, and sib-combined designs and at 0.188 for the sib-radial design, as would be presumably be done if DNA is pooled once then used repeatedly in a genome- wide screen of markers.
  • the allele frequency is varied for a phenotype with a dominant inheritance (Fig. 5 A), additive inheritance (Fig. 5B), and recessive inheritance (Fig. 5C) with respect to the minor allele.
  • the population size is rather insensitive to allele frequency for/? > 0.01 for dominant and additive inheritance, and for/? > 0.2 for recessive inheritance, for all but the sib-radial design, indicating that the analytic theory is valid in these regions.
  • the population size required to detect association increases rapidly as the allele frequency decreases below these limits.
  • the sib-radial design is more sensitive to the allele frequency than the other designs, losing power rapidly as the allele frequency falls below 0.1 for dominant and additive inheritance and 0.2 for recessive inheritance.
  • the sib-radial design is an exception, with increasing requirements only for strong over-dominance.
  • d/a -2.
  • Verkasalo PK Kaprio J, Koskenvuo M, Pukkala E (1999) Genetic predisposition, environment and cancer incidence: a nationwide twin study in Finland, 1976-1995. Int J Cancer 83: 743-749.

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Abstract

L'identification des composants génétiques de maladies complexes représente un des buts principaux du projet génome humain. Ces maladies et leurs facteurs de risque sous-jacents sont souvent mieux décrits par des phénotypes quantitatifs que par une distinction arbitraire entre des individus atteints ou non atteints de telles maladies. Des études d'association permettent d'identifier des loci génétiques participant directement dans ces loci de caractères quantitatifs (QTL), mais nécessitent une vaste population. Des études de populations de paires d'enfants de mêmes parents ont été proposées en vue d'accroître l'efficacité de telles études dans le cas où des populations sont stratifiées et des essais effectués sur des ADN mis en commun qui peuvent contribuer à réduire la charge expérimentale, mais ces approches ont été analysées surtout dans le cas des phénotypes atteints/non atteints de maladies. L'invention concerne des procédés efficaces permettant de cartographier des QTL au moyen d'ADN mis en commun à partir de paires d'enfants de mêmes parents. Un essai préféré mettant en oeuvre un ensemble unique de mises en commun permet de sélectionner des enfants de mêmes parents qui n'ont pas de relation au moyen de valeurs phénotypiques extrêmes, nécessitant une population approximativement 1,5 fois supérieure à celle destinée à un génotypage individuel. Une stratégie globale préférée, nécessitant une population seulement 1,24 fois supérieure à celle destinée à un génotypage individuel, consiste en un essai combiné d'ADN mis en commun selon des valeurs phénotypiques moyenne et de différence entre des enfants de mêmes parents. La fraction optimale de mise en commun est de 27% et n'est pas sensible à tous les paramètres de modèle, notamment la fréquence des allèles, le mode de transmission héréditaire et l'amplitude de l'effet des QTL.
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